Leptin (OB, the product of ob gene), is a pleiotropic molecule mainly secreted by white adipocytes that plays a relevant role in the regulation of body weight and food intake (for Review see Gonzalez et al., 2000). In contrast to leptin, the leptin receptor (OB-R, the product of the db gene) has several spliced variants. The full-length and functional OB-R (OB-Rb) is expressed by the hypothalamus and plays a key role in the energy balance process (Tartaglia et al., 1995). OB-R isoforms with shorter cytoplasmatic tail are expressed in many tissues, but their function remain unknown (Wang et al., J. Biol. Chem., 272:16216-16223 (1997)). A soluble OB-R that could regulate leptin biological actions has been also described (Lewandowski et al., J. Clin. Endocrinol. Metab. 84: 300-306 (1999)). Leptin sequence is highly conserved in many species but some differences in OB-R sequences are found between species.
Binding of leptin to OB-R induces the homodimerization of the receptor that in turns allow the binding of Janus kinase 2 (JAK2) to specific box motifs in the intracytoplasmatic tail of OB-R. JAK2 phosphorylates OB-R followed by phosphorylation of signal transducer and activator of transcription 3 (STAT3), which, in turn dimerizes and translocates to the nucleus, thus activating several downstream signaling pathways. In addition to the JAK-STAT signaling pathway, other pathways, including the mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and PI3-kinase pathways, are also activated by leptin (Ghilardi et al., Proc. Natl. Acad. Sci. USA 93: 6231-6235 (1996); Zabeau et al., FEBS Lett. 546: 45-50 (2003)).
Since the discovery of leptin 10 years ago (Zhang et al., 1994) emerging evidence have been accumulating that strongly link leptin signaling with the reproductive function (for review see Gonzalez et al., 2000; Castracane and Henson, Semin. Reprod. Med. 20 (2): 89-92 (2002)).
In vitro studies have shown that leptin and OB-Rb are expressed by female reproductive tissues, including ovaries (Cioffi et al., Mol. Hum. Reprod. 3: 467-472 (1997); Zachow et al., Endocrinol. 138: 847-850 (1997); Agarwal et al., J. Clin. Endocrinol. Metab. 84: 1072-1076 (1999)), oocytes, preimplantation embryo (Matsuoka et al., Biochem. Byophys. Res. Commun., 256: 480-484 (1999); Kawamura et al., Endocrinology 143:1922-1931 (2002)), endometrium (Gonzalez et al, 2000; Wu et al., Mol. Hum. Reprod. 8: 456-464 (2002)) and placenta (Masuzaki et al., Nat. Med. 3:1029-1033 (1997); Senaris et al., Endocrinol. 138: 4501-4504 (1997)). Leptin can promote the development of mouse preimplantation embryos through OB-R signaling (Kawamura et al., Endocrinology 143:1922-1931 (2002)). Leptin protein has been found in human and mouse oocytes, and preimplantation embryos (Cioffi et al., Mol. Hum. Reprod. 3: 467-472 (1997); Antczak and Van Blerkom, Mol. Hum. Reprod. 3: 1067-1086 (1997)). But, leptin mRNA has been only found at blastocyst stage. Leptin at physiological concentrations could positively affect phosphorylation of STAT3 (p-STAT3) in mouse oocytes (Matsuoka et al., Biochem. Biophys. Res. Commun. 256: 480-484 (1999)). These data suggest that OB-R signaling in oocytes and early preimplantation embryos up to morula stage would require maternal supply of leptin (Kawamura et al., Endocrinology 143: 1922-1931 (2002)). A particular cell-borne pattern for leptin and STAT3 has been found in outer blastomers of human and mouse blastocysts (Antczak and Van Blerkom, Mol. Hum. Reprod. 3: 1067-1086 (1997)). These data would indicate that leptin/OB-R are required to establish the cross-talk between the implanting embryo and the receptive endometrium.
In vitro the secretion of leptin is regulated by human preimplantation embryos co-cultured with endometrial cells (Gonzalez et al., 2000). Leptin induces the acquisition of the invasive phenotype of human trophoblast cells (Castellucci et al., Mol. Hum. Reprod. 6 (10): 951-8 (2000); Gonzalez et al., Early Preg. Biol. Med. 5:132-143 (2001)). Leptin increase the levels of β3-integrin (a marker of endometrial receptivity) in human endometrial epithelial cells (Gonzalez and Leavis, 2001). Moreover, leptin in a dose-dependent manner increased p-STAT3 and leukemia inhibitory factor (LIF), interleukin-1 (IL-1) and levels of their cognate receptors in rabbit (Gonzalez and Leavis, 2003) and human endometrial cells (Gonzalez et al., 2004). Lastly, blockade of the OB-R with antibodies abrogated leptin-induced effects suggesting that leptin signaled through OB-R and the JAK/STAT3 pathways (Gonzalez et al 2003; Gonzalez et al., 2004).
Although the specific mechanisms whereby leptin modulates reproductive function are not completely understood leptin appears to be essential for normal preimplantation and/or implantation processes. Overall in vitro and in vivo data suggest that leptin signaling impact implantation capabilities in both entities: preimplantation embryo and endometrium.
In vivo studies have shown that mouse mutant deficient in leptin (ob/ob) (Zhang et al., 1994) or OB-R (db/db) are obese and infertile. Fertility can be restored in ob/ob by exogenous leptin (Chehab et al., Nat. Genet. 12: 318-320 (1996)). The withdrawal of leptin infusion in ob/ob females short after fertilization impairs implantation (Malik et al., Endocrinology 142: 5198-5202 (2001)). Leptin injection into starved mice restores fertility (Ahima et al., Nature 382: 250-252 (1996)). A postovulatory increase in serum leptin concentration appears to be associated with implantation potential (Cioffi et al., Mol. Hum. Reprod. 3: 467-472 (1997)) and low expression of OB-R has been found in endometrium from women with unexplained infertility (Alfer et al., Mol. Hum. Reprod. 6: 595-601 (2000)). These data suggest that in vivo leptin could act in an autocrine or paracrine manner to regulate biological functions that may mediate the implantation process.
From the analysis of a structural-based model for leptin/OB-R complex the helices I and III of leptin are likely the interacting regions for its binding to OB-R (Gonzalez and Leavis, 2003). In consequence, a peptide (LPA-2) derived from helix III of leptin is able to inhibit leptin binding to its receptor in vitro. Moreover, LPA-2 interferes with the leptin signaling pathways responsible for leptin-induced increase in levels of IL-1, LIF and β3-integrin by rabbit and human endometrial cell cultures (Gonzalez and Leavis, 2003; Gonzalez et al., 2004).
These data suggest that targeting the leptin receptor may negatively affect implantation. Therefore, it was hypothesized that the inhibition of OB-R function (in endometrium and/or preimplantation embryos) by LPA-2 or anti mouse OB-R antibodies will impair mouse embryo implantation. In the present study, the impact of the intrauterine injection of LPA-2 and anti-OB-R antibodies at Day 3 of pregnancy in a mouse model was investigated. Both OB-R inhibitors impaired mouse implantation and affected the endometrial expression of several molecules related to the implantation potential. Overall, our results suggest that leptin could be one of the primary factors that initiates and regulates the cascade system of molecules that promote the development of endometrial receptivity and successful implantation.